The maintenance of intracellular calcium (Ca) levels requires the fine regulation of numerous calcium handling proteins. Voltage-gated Ca channels define a primary path of Ca entry, which then influences a host of vital functions. Of the two main classes of Ca channels found in the cardiovascular system, L-type and T-type, surprisingly little is known about the functions of the latter. The broad, long-term objective of this proposal is to understand the role of T-type Ca channels in the cardiovascular system, with particular focus on instances where they appear to predominate and have unique functions over high voltage-activated, L-type channels. The Specific Aims of this project investigate two main areas of cardiovascular physiology: myocyte proliferation (both cardiac and vascular smooth muscle), and the development of cardiac contractility. Many independent lines of evidence indicate cell cycle-specific regulation of T-type Ca channels and a possible role in proliferation; however, more studies are needed develop rational hypotheses for the mechanisms of T-channel influence. Molecular biology techniques will be used to manipulate T-type Ca channel expression, using primary cultures of either developing cardiomyocytes or proliferating arterial vascular smooth muscle cells. The consequences will be studied in relation to cell proliferation, to better define a role for LVA Ca entry. Cardiomyocyte proliferation is an important aspect of cardiac development, and understanding the mechanisms may have relevance for timely research on cardiac regeneration and repair. Proliferation studies on VSM are relevant for pathological vascular remodeling, an actively pursued target of potential gene therapy protocols. In a second Specific Aim, the contributions of T-type Ca channels to early cardiac contractility will be defined using embryonic stem cell lines that differentiate into cardiomyocytes. T-type Ca channels will be eliminated in undifferentiated ES cells by gene targeting, and channels will be over-expressed using adenoviral vectors or by creating stable transfected ES cell lines. The consequences of loss- and gain-of-function for development and properties of automatic contractile function will be assessed by electrophysiology and calcium imaging protocols. These studies make use of the most current gene transfer, Ca imaging and electrophysiology techniques to ask focused, important questions about an understudied class of Ca channels.